5

# ATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAG CAAC TGAAAGCT 'GTTATGGATGATTTCGCAGCTZTTGTAGAGAAGTGCTGCAAGGCTGAC GATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAA...

## Question

###### ATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAG CAAC TGAAAGCT 'GTTATGGATGATTTCGCAGCTZTTGTAGAGAAGTGCTGCAAGGCTGAC GATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCC TTAGGCTTATAACATCACATTTAAAAGCATCTCAGCCTACCATGAGHATAAGAGAAAGAA AATGAAGATC4ALAGCTTATTCATCTGTITTTCTTTZTCGTTGGTGTAAAGCCAACACCC TGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAAT AAAAAATGGAAAGAATCTAATAGACTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGC TATCCTGAAAAZTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGG TAGA

ATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAG CAAC TGAAAGCT 'GTTATGGATGATTTCGCAGCTZTTGTAGAGAAGTGCTGCAAGGCTGAC GATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCC TTAGGCTTATAACATCACATTTAAAAGCATCTCAGCCTACCATGAGHATAAGAGAAAGAA AATGAAGATC4ALAGCTTATTCATCTGTITTTCTTTZTCGTTGGTGTAAAGCCAACACCC TGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAAT AAAAAATGGAAAGAATCTAATAGACTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGC TATCCTGAAAAZTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGG TAGAGGETTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAAAAAA AZAAAAAALAAAAAAGCCGAATTCTGCAGATAZCCATCACACTGGCGGCCGCTCGAGCAT GCATCTAGAGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTT TTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCCTCAGAGATCGATCGATCGATTG TTGCATGAGAAAACGCCAGTAAGTGACAGACTCACCAAATGCTGCACAGAATCCTTGGTG AACACCCGACCATGCTTTTCAGCTCTGGAAGTCCATGAAACATACGTTCCCAAAGAGTTT AATGCTGAAACATTCACCTTCCATGCAGATATAPGCACACTTTCTGAGAAGGAGAGACAA ATCAAGAAACAAACTGCACTTGTTGCAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAG CZACTGAAAGCTGTTATGGH TGATTTCGCAGCTTZTGTAGAGAAGTGCTGCAAGGCTGAC GATAA GGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTIGTTGCTGCAAGTCAAGCTGCC ITAGGCTTATAACATCACATTTAAAAGCATCTCACCCTACCATGAGAATAAGAGAAAGAA la) Using the sequence above, design your forward and reverse primers; following the guidelines Primer length 15-30 nucleotides Tm should range between 55 65C Tm of primer pair should be within SC of each other GC content of primer = 40 60% Try to avoid a row of 4 or more ofthe same bases s0 for eg: GGGGG or ATATATATAT 3' end of primer should end with G or â‚¬ 1b) Provide the sequences in a 5'-3' direction as if you were going to order them: Include the start and stop bases and number for both primers and length of primer. Forward primer in 5' 3' direction. Reverse primer in 5' 3' direction Ic) Estimate the melting temperature of the primers You designed Tm =4(number of GC) + 2(number of AT) 1d) Describe the thermal cycling protocol that you would predict to work Provide the denaturation temperature, the 'annealing temperature, and the extension temperature:

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